Means for identifying the locus of a major resistance gene to the rice yellow mottle virus, and their applications

ABSTRACT

The invention relates to a method for identifying the markers of the locus of a major resistance gene with respect to RYMV. The invention method comprises the following steps: selective amplification of fragments of rice DNA from resistant individuals and sensitive individuals descending from parental varieties, whereby said fragments undergo a prior digestion phase followed by ligation in order to fix additional initiators having one or more specific nucleotides at the extremities thereof; separation of the amplification products; comparison of the electrophoresis profiles obtained by mixing fragments from resistant descendants and sensitive de4scendants with fragments from parental varieties, in order to identify strips where polymorphism is genetically linked to the resistance locus. Said identification is followed by a validation step in which verification occurs on all individuals and the genetic recombination rate between the marker and the resistance locus is calculated. The invention can be used to identify resistant phenotypes and transfer the RYMV resistance gene.

This application is a continuation-in-part of PCT Application No.PCT/FROO/01742, filed Jun. 21, 2000, which designated the U.S., theentire content of which is incorporated herein by reference.

[0001] The invention relates to the means, tools and methods foridentifying the locus of a major resistance gene to the Rice YellowMottle Virus (RYMV) in short. It more particularly relates to markersand PCR primers in respect of tools.

[0002] RYMV is a virus that is endemic in Africa. In a few rarevarieties of the African species of cultivated rice Oryza glaberrima, avery high resistance to RYMV has been identified. But since theinterspecific hybrids between the two species of cultivated rice areextremely sterile, prior research has not been able to describe eitherthe genetic bases or the mechanism of this resistance.

[0003] Research by the inventors in this area has shown that a varietycalled Gigante which originated from Mozambique and was identified byADRAO, and which is a member of the cultivated Asian rice species Oryzasativa, shows the same characteristics as those observed with O.glaberrima. The inventors have characterized RYMV resistance bydemonstrating that it is related to a major recessive resistance genethat is identical in both sources of resistance under consideration (O.Sativaand O. glaberrima).

[0004] This resistance occurs at the level of cell-to-cell movement andleads to blockage of the virus at the infected cells whereas virusreplication is normal.

[0005] On the contrary, in resistant varieties, a mutation of saidprotein does not enable anymore the association with the virus, and thusits diffusion in the plant.

[0006] The migration of RYMV occurs under the form of a mucleoproteiccomplex associating viral nucleic acid, of protein and movement virusprotein. In the cells of sensitive varieties, an host factor, profably aprotein, also contributes to the movement of the virus.

[0007] Having regard to these results, the inventors prepared specificmethod and tools for characterizing the chromisome fragment bearing saidresistance to RYMV gene which codes for said protein enabling themovement of the virus in the plant.

[0008] The purpose of the invention is therefore to provide a method foridentifying molecular markers of the resistance locus to RYMV.

[0009] It also concerns the DNA fragments such as revealed by thismethod and which can be used as markers.

[0010] The invention also concerns applications of such markers, inparticular to define other markers having high specificity to theresistance locus and to predict a resistant phenotype.

[0011] The invention further relates to sequences of primers, as newproducts, used in the PCR techniques applied.

[0012] According to the invention, the identification of markers of thelocus of a major resistance gene to RYMV, comprises the use of AFLPmarkers (Amplified Fragments Length Polymorphism) and uses the PCRtechnique.

[0013] This method of identification is characterized in that itcomprises:

[0014] selective amplification of rice DNA fragments firstly fromresistant individuals and secondly from sensitive individuals,descending from parent varieties, these fragments being previouslysubmitted to a digestion step, followed by ligation to fix complementaryprimer adapters having, at their end, one or more specific nucleotides,one of the primers in the primer pair being labelled for developmentpurposes,

[0015] separating the amplification products by gel electrophoresisunder denaturing conditions, and

[0016] comparing the electrophoresis profiles obtained with mixtures offragments derived from resistant descendants and with mixtures derivedfrom sensitive descendants, with the fragments derived from parentvarieties, for the purpose of identifying bands whose polymorphism isgenetically linked to the resistance locus, this identification beingoptionally followed, for validation purposes, by verification on each ofthe individuals and by calculation of the genetic recombination ratebetween the marker and the resistance locus.

[0017] In one embodiment of the invention, the DNA fragments areobtained by digestion of the genomic DNAs of resistant plants and ofsensitive plants, and their parents, using restriction enzymes.

[0018] Restriction enzymes which have proved to be suitable includeEcoRI and MseI.

[0019] Short nucleotide sequences are fixed to digestion fragments(adapters) to generate blunt ends to which the adapters are subsequentlyfixed.

[0020] The primers used in the amplification step are complementary tothese adapters with, at their 3′ end, from 1 to 3 nucleotides which maybe variable.

[0021] The amplification step is advantageously conducted using the PCRtechnique.

[0022] Specific amplification profiles are obtained with primer pairsrespectively having AAC and CAG, ACC and CAG motifs at their end, orfurther AGC and CAG.

[0023] The sequences corresponding to the EcoRI and MSeI adapters arerespectively GAC TGC GTA CCA ATT C (SEQ ID N^(o) 1) and GAT GAG TCC TGAGTA A (SEQ N^(o) 2).

[0024] The primer pairs used for amplification are then advantageouslychosen from among E-AAC/M-CAG; E-ACC/M-CAG; and E-AGC/M-CAG; in which Eand M respectively correspond to SEQ ID N^(o) 1 and SEQ ID N^(o) 2.Other pairs are given in table 6 in the examples.

[0025] Comparative study of the amplification profiles obtained revealspolymorphic bands specifically present in the sensitive varieties andtheir sensitive descendants, as shown in the examples, and consequentlycorresponding to resistance markers.

[0026] In particular, development by gel electrophoresis underdenaturing conditions leads to identifying 2 marker bands M1 and M2 ofrespectively 510 bp and 140 bp.

[0027] According to analysis of segregation data, these 2 bandsdetermine a chromosome segment of 10 to 15 cM carrying the resistancelocus and are located either side of this locus at 5-10 cM.

[0028] According to one provision of the method of the invention, thepolymorphic bands identified as markers specific to the RYMV resistancelocus, are isolated from gels. Advantageously the electrophoresis gelsare excised. This isolation step is followed by purification usingconventional techniques. In this manner DNA fragments are obtained.

[0029] According to another provision of the invention, said purifiedfragments are cloned in an appropriate vector, such as a plasmid,inserted into the host cells, in particular bacterial cells such asthose of E. coli.

[0030] According to another provision of the invention, the purified,cloned DNA fragments are sequenced.

[0031] Taking advantage of the sequences of the inserts corresponding tosaid DNA fragments, the invention also provides a method for obtainingmarkers having high specificity for the locus of a major resistance geneto RYMV. This method is characterized in that PCR primer pairs aredetermined which are complementary to the fragments of the sequence of agiven insert, specific amplification of the insert is made using theseprimer pairs, and the amplification products are then subjected tomigration on electrophoresis gel.

[0032] These DNA sequences can be used to identify a polymorphism linkedto the resistance locus in a rice variety to be examined using differentmethods as described in the examples:

[0033] 1) by directly identifying a size polymorphism of these DNAsequences after specific amplification and separation of the fragmentson agarose gel,

[0034] 2) by digesting the amplification products with restrictionenzymes to separate the digestion products on agarose gel,

[0035] 3) by using these sequences as probes to hybridize the DNA ofrice varieties previously digested by a restriction enzyme and todetermine a restriction polymorphism.

[0036] The invention concerns, as new products, the polymorphous AFLPbands such as identified by the method defined above, from the DNA ofrice plants, optionally isolated, purified and sequenced.

[0037] These AFLP bands are characterized in that they are specificallyrevealed in a variety sensitive to RYMV (IR64) and in the fraction ofsensitive plants derived from the crossing of this variety with theGigante resistance variety as described in the examples.

[0038] The invention particularly concerns the DNA sequencescorresponding to these polymorphous bands, which can be used to define asegment of chromosome 4 of 10-15 cM carrying the resistance locus toRYMV.

[0039] Having regard to the method with which they are obtained, theAFLP bands correspond to restriction fragments and in particular,according to one embodiment of the method of the invention, toEcoRI-MseI fragments.

[0040] Fragments of this type are called M1 and M2 markers and arecharacterized by a size, of 510 bp and 140 bp respectively, inelectrophoresis gel under denaturing conditions.

[0041] These fragments are characterized in that they correspond to DNAsequences flanking the resistance locus and located either side of thelatter at 5-10 cM.

[0042] The invention also concerns fragments cloned in vectors such asplasmids, these cloning vectors as such, characterized in that theycomprise such fragments, and the host cells transformed using thesevectors, such as bacterial cells, for example E. coli.

[0043] The invention relates in particular to the DNA sequencecorresponding to the fragment identified as M1 marker and meeting thefollowing sequence SEQ ID N^(o) 3:CGTGCTTGCTTATAGCACTACAGGAGAAGGAAGGGGAACACAACAGCCATGGCGAGCGAAGGTTCAACGTCGGAGAAACAGGCTGCGACGGGCAGCAAGGTGCCGGCGGCGGATCGGAGGAAGGAAAAGGAGGAAATCGAAGTTATGCTGGAGGGGCTTGACCTAAGGGCAGATGACGAGGAGGATGTGGAATTGGAGGAAGATCTAGAGGAGCTTGAGGCAGATGCAAGATGGCTAGCCCTAGCAACAGTTCATACGAAGCGATCGTTTAGTCAAGGGGCTTTCTTTGCGAGTATGCGCTCAGCATGGAACTGCGCGAAAGAAGTAGATTTCAGAGCAATGAAAGACAATCTGTTCTCGATCCAATTCAATTGTTTGGGGGATTGGGAACGAGTTATGAATGAAGGTCCATGGACCTTTCGAGGATGTTCGGTGCTCCTCGCAGAATATGATGGCTGGTCCAAGATTGAAT

[0044] The DNA sequence of the Ml marker has a size of 471 bp.

[0045] The invention also concerns, as new products, the sequences ofnucleotides used as PCR amplification primers.

[0046] Such primers comprise the pairs E-AAC/M-CAG; E-ACC/M-CAG;E-ACC/M-CAG; in which E and M respectively relate to SEQ ID N^(o) 1 andSEQ ID N^(o) 2.

[0047] Other primers are complementary to sequences identified in thesequence of the fragment designated by marker M1. These are inparticular (5 ′,3′) sequences chosen from among: AGGAAGGGGAACACAACAGCC(21 bp) (SEQ ID NO 4) TTATGCTGGAGGGGCTTGACC (21 bp) (SEQ ID NO 5)GCAGTTCCATGCTGAGCGCAT (21 bp) (SEQ ID NO 6) CCGAACATCCTCGAAAGGTCC (21bp) (SEQ ID NO 6) TCATATTCTGCGAGGAGCACC (21 bp) (SEQ ID NO 8)

[0048] The invention also concerns the DNA sequence corresponding to thefragment identified as marker M2 and corresponding to sequence SEQ IDN^(o) 9 AATTCACCCC ATGCCCTAAG TTAGGACGTT CTCAGCTTAG TGGTGTGGTAGCTTTTTCTA TTTTCCTAAG CACCCATTGA AGTATTTTGC ATTGGAGGTG GCCTTAGGTTTGCCTCTGTTA

[0049] The size of M2 is 120 bp.

[0050] Specific primers complementary to sequences identified in thesequence of M2 were defined. Said sequences meet the followingsequencing (5′,3′): AACCTAAGGCCACCTCCAAT SEQ ID NO 10GCAAACCTAAGGCCACCTC SEQ ID NO 11 ATTCACCCCATGCCCTAAG SEQ ID NO 12

[0051] According to a further aspect of the invention, the latterconcerns the use of DNA sequences obtained with the above primers todefine polymorphisms which can be used to identify resistant phenotypes.

[0052] The invention also concerns a method for identifying the DNAsequence carrying the major resistance gene to RYMV. This method ischaracterized by screening a bank consisting of DNA fragments of 100 to150 kb of the IR64 or other variety, such as the BAC bank (BacterialArtificial Chromosomes) cloned in bacteria, to select the clone orclones from the bank containing the markers defined above and theresistance gene to RYMV.

[0053] This type of BAC bank is available from the IRRI institute.

[0054] The existence of different restriction sites on the sequencecorresponding to the M1 marker, and in particular the sitescorresponding to HpaII/Mspl, provides for advantageous identification ofresistant phenotypes.

[0055] The identification of different restriction sites on the sequencecorresponding to the M1 marker enables characterization of apolymorphism which may be put to advantageous use to map the M1 markeron rice genetic linkage maps.

[0056] The map of the sequence corresponding to the M1 marker can beused to identify a chromosomal zone on chromosome 4 of rice carrying theRYMV resistance locus.

[0057] The map of the RYMV resistance gene on chromosome 4 of the ricegenetic map allows identification of the markers the closest to theresistance locus. These are in particular the microsatellite markersRM252 and RM273 or any other marker inside the (4-5cM) space defined bythese markers allowing identification of a polymorphism between the IR64and Gigante parents, such as the RFLP markers screened from genomicbanks or cDNA, microsatellites, AFLP markers or markers derived fromphysical mapping of the region such as BAC, YAC clones or their cosmids.

[0058] The markers identified in accordance with the invention, or anyother marker located in this space allowing identification of apolymorphism between resistant varieties such as Gigante or O.Glaberrima with RYMV-sensitive rice varieties, may be used for transferof RYMV resistance into sensitive varieties by successive backcrossesfollowed by marker-assisted selection.

[0059] Other characteristics and advantages of the invention will begiven in the following examples, in which reference is made to FIGS. 1to 10 which respectively represent:

[0060]FIG. 1: cloning of marker M1 in the PGEMTeasy plasmid. Digestionof the plasmid shows a DNA fragment of 510 bp corresponding to band M1;

[0061]FIG. 2: amplification of marker Ml in the four rice varieties(Azucena, Gigante, IR64 and Tog5681) using the primer pairs (2-4): 291bp; (2-5): 310 bp; (1-3): 288 bp; (1-4): 406 bp; (1-5): 425 bp; (2-3).The M1 fragment is slightly bigger in Tog5681 than in the othervarieties;

[0062]FIG. 3: identification of restriction sites on the sequence of theM1 marker in the 4 varieties IR64, Azucena, Gigante and Tog5681;

[0063]FIG. 4: digestion of the Ml marker with the HpaII enzyme after PCRamplification using primer pairs (1-3), (1-4) and (1-5)on the fourvarieties (Azucena, Gigante, IR64 and Tog5681). The presence of a HpaIIrestriction site in the IR64 and Tog568 varieties releases a fragment of86 bp which reduces the size of the amplified fragment to the sameextent.

[0064]FIG. 5: characterization of the M1 marker on sensitive andresistant plants of F2 issue(IR64 and Gigante). The resistant F2 plantshave the profile of the resistant parent (IR64 - no Hpall site), withthe exception of a single recombinant, the resistant plants have theprofile of the sensitive parent (IR64-presence of HpaII site) with theexception of two recombinants;

[0065]FIG. 6: segregation of the M1 marker in the HD population(IR64×Azucena); IR64-Azucena-30 HD individuals (IR64×Azucena);

[0066]FIG. 7: the genetic linkage map of chromosome 4 of rice with thepositioning of marker M1 and identification of the space interval inwhich the resistance locus is found;

[0067]FIG. 8: hybridization of M1 marker used as probe on membranescarrying the DNA of the 4 varieties (IR64, Azucena, Gigante and Tog5681) digested by 6 restriction enzymes ApaI, KpnI, PstI, Scal, HaeIII.The Tog5681 variety shows a different restriction profile to the othervarieties for the Scal enzyme which may be used to label the resistancelocus of this variety; and

[0068]FIG. 9: hybridisation of the M1 marker used as probe on membranescarrying the DNA of individuals derived from backcross (IR64×Tog568)×Tog 5681 and digested with the Scal enzyme. These descendants are insegregation for RYMV resistance. The sensitive individuals (5) all showthe IR64 band associated with the Tog5681 band (heterozygoteindividuals). The resistant individuals (9) only show the Tog5681 bandwith the exception of one recombinant individual,

[0069]FIG. 10: mapping and anchoring of the locus of bred resistance toRYMV on the map IR64×Azucena, and

[0070]FIG. 11, the genetic map of the region flanking the resistancegene in the IR64×Gigante population (figure 11A) and the simplifiedrepresentation of contig 89 and of part of the clones assigned to thiscontig.

EXAMPLE 1 Identification of resistant-source varieties

[0071] The varieties used in the resistance study, and especially thetwo resistant varieties Gigante and Tog5681, were characterized usingmicrosatellite markers on a representative sampling of loci.

[0072] Polymorphism is evidenced by the number of repeats of a shortnucleotide pattern, most often binucleotide which is characteristic of agiven variety.

[0073] On a set of loci, the catalogued alleles can provide specificcharacteristics for each variety.

[0074] The detection of these microsatellite markers is made by DNAamplification using the specific primers determined by Chen et al (1)followed by migration on polyacrylamide gel under denaturing conditionsin accordance with the protocol described by the same authors.

[0075] Table 1 gives the results using a reference system drawn up byChen et al above, according to which the alleles are identified by thenumber of pattern repeats compared with the IR36 variety used ascontrol. The two varieties Gigante and Tog5681 are thereforespecifically described on 15 loci in respect of any other varieties (themicrosatellite markers are given in column one). TABLE 1 Size on LocusChr IR36 Ref. IR36 Gigante IR64 Azucena Tog568113 RM001 1 113 (2) n n −26 n n − 22 n − 26 RM005 1 113 (2) n n − 6 n − 4 n + 16 n − 8 RM011 7140 (2) n n − 4 n n − 24 n − 16 RM018 7 157 (2) n n + 4 n + 6 n + 8 n −6 RM019 12 226 (2) n n n + 21 n − 9 n − 21 RM021 11 157 (2) n n + 8 n n− 14 n − 32 RM148 3 129 (3) n n + 6 n n n + 6 RM167 11 128 (3) n n + 4 nn + 32 n + 24 RM168 3 116 (3) n n − 20 n n − 20 n − 24 RM232 3 158 (1) nn − 14 n n − 12 n − 16 RM022 3 194 (2) n n − 2 n n − 4 n − 2 RM252 4 216(1) n n + 38 n + 2 n − 20 n + 10 RM255 4 144 (1) n n n n n RM246 1 116(1) n n − 12 n − 12 n − 16 n − 12 RM231 3 182 (1) n n + 6 n − 22 n − 4 n− 12

EXAMPLE 2 Characterization of resistance

[0076] Resistance was characterized using artificial inoculation ofyoung seedlings with the virus, compared with an extremely sensitivecontrol variety IR64.

[0077] The virus content was followed up for 60 days after inoculationusing ELISA tests on the most recent leaves.

[0078] These tests were never able to demonstrate a signal that wassignificantly different to the signal of control plants non-inoculatedwith the virus.

[0079] A further experiment was conducted by inoculating isolatedprotoplasts of the two varieties Tog5681 and Gigante. In both cases, itwas possible to detect the presence of viral proteins (capsid proteinand P1 movement protein) and the accumulation of viral DNA,demonstrating the capacity of these protoplasts to multiply the virus,in the same manner as the protoplasts of sensitive varieties such asIR64.

[0080] Therefore, if it is considered that replication, cell-to-cellmovement and long-distance transport through the vessels are the threemain steps in the process of the infectious cycle within the plant, theresistance of these two varieties most logically lies in blockage of thevirus at the infected cells.

EXAMPLE 3 Resistance genetics

[0081] Different F1 crosses were made between the resistant O. sativavariety (Gigante), a resistant O. glaberrima variety (Tog5681- alsoidentified by ADRAO), and the highly sensitive control variety IR64(selected at the IRRI).

[0082] Culture of the plant material, crosses and production ofdescendants were made in the IRD greenhouses in Montpellier.

[0083] The F1 hybrids obtained between the sensitive and resistantvarieties were tested for resistance to the RYMV virus by ELISA testingand follow-up of symptoms.

[0084] These F1 hybrids proved to be as sensitive as the sensitiveparent, and therefore showed that that the type of resistance isrecessive.

[0085] On the other hand, the hybrids between the two resistance sourcesGigante and Tog5681 only yielded resistant F1 hybrids to the benefit ofa single resistance locus in these sources of resistance.

[0086] These results are summarized in Table 2 below.

[0087] This table gives the distribution of ELISA responses (A 405 nm)in the leaves infected by systemic route of Fl hybrids, of backcrossesand of F2 descendants obtained from backcrosses between the sensitiveIR64 variety and the 2 resistant cultivars Gigante and Tog5681. TABLE 2Average Presence Number of Distribution of OD values values F1 hybriddescendants of symptoms genotypes (0.01-0.05) (0.9-1) >1 OD Derivativesof Tog5681 F1: (IR64 × Tog 5681) Sensitive — — — 10 1.9 BCS: (IR64 × Tog5681) × IR64 Sensitive 19  6 4 15 1.6 BCS: (IR64 × Tog5681 c Tog5681 Insegregation 22 12 — 10 — Derivatives of fertile BCS plant BCS F2Sensitive 11 — — 11 1.3 BCS × IR64 Sensitive  1 — —  1 1.9 BCS × Tog5681sensitive 15 — — 15 1.9 Gigante derivatives — — — F1 (IR64 × Gigante) —— — 10 1.9 F2: (IR64 × Gigante) In segregation 65 15 — 50 — F1: (Gigante× Tog5681) Sensitive — 10 — — 0.3

[0088] In respect of Gigante, the heredity of resistance was confirmedby a resistance test on 55 F3 families resulting from the cross between(IR64×Gigante) . The results are given in Table 3.

[0089] This table gives the segregation of RYMV resistance in F3descendants (IR64×Gigante). Inoculation was made 10 to 17 days aftergermination with the Burkina Faso isolate and symptoms were followed upfor 45 days after inoculation. TABLE 3 Classes of Number of Number ofplants resistance descendants Total Sensitive Resistant Incidence ofresistant plants Sensitive 15 191 191 0 0 In segregation 30 343 262 010.24 2 = 0.07 (3:1) Resistant 4 45 14 31 0.69 Very resistant 6 87 0 87 1Resistant* 7 73 23 50 0.60 Very resistant* 4 56 0 56 1

[0090] EXAMINATION OF THIS TABLE SHOWS THAT

[0091] ¼ of F2 plants only give resistant plants in F3 descendants, andare homozygote for resistance,

[0092] ¼ of F2 plants only give sensitive plants in F3 descendants, andare homozygote for sensitivity,

[0093] ½ of F2 plants are in segregation for resistance and givesensitive and resistant plants in the same proportion (3:1) in F3descendants.

[0094] All these results tally perfectly with a single recessiveresistance gene occurring in the two varieties Gigante and Tog5681.

EXAMPLE 4 Identification M1 and M2 resistance markers using the AFLPprotocol

[0095] A - OBTAINING DNA POOLS

[0096] The leaves of 10 sensitive plants and 10 resistant plants derivedfrom an F2(IR64×Gigante) were sampled for their DNA extraction.

[0097] The DNA were then mixed stoechiometric fashion to form two DNApools respectively corresponding to 10 sensitive or resistant F2 plantsand with a final mixture concentration of 50 ng/μ l. These mixturesserved as basis for the identification of resistance markers using theAFLP (Amplified Fragments Length Polymorphism) method developed byZaneau et al (4) and Vos et al (5). The products used are in the form ofa commercial kit (Gibco BRL) available from Keygene & Life Technologies.

[0098] b - Obtaining restriction fragments

[0099] 250 ng of each of the DNA pools at 50 ng/μ l and of the parentsare digested simultaneously by two restriction enzymes (EcoRI and MseI).

[0100] Digestion reaction (25 μl)

[0101] 5 μl DNA (50 ng/ml)

[0102] 0.2 μl (2U) EcoRI (10U/μl)

[0103] 0.2 μl (2U) MseI (5U/μl)

[0104] 5 μl 5X T4 ligase buffer

[0105] 14.5 μl H₂ O

[0106] The digestion reaction is carried out for two hours at 37° C.,then for 15 min at 70° C. to inactivate the restriction enzymes. Afterdigestion, the ligation reaction was performed.

[0107] Ligation reaction (50 μl):

[0108] 25 μl double digestion reaction medium

[0109] 1 μl EcoRI adapter

[0110] 1 μl MseI adapter

[0111] 5 μl 5X T4 ligase buffer

[0112] 1 μl (1 U) ligase (10U/μ l)

[0113] 17 μl H₂ O

[0114] The ligation reaction is conducted at 37° C. for 3 hours followedby inactivation of the enzyme at 60° C. for 10 min.

[0115] c - Amplification

[0116] Amplification properly so-called was performed in two steps:preamplification and specific amplification.

[0117] c1- Preamplification reaction (50 μl)

[0118] 5 μl of reaction medium containing the digested DNA fixed to theadapters, diluted to {fraction (1/10)} 0.5 μl EcoRI primer (150 ng/μl)

[0119] 2 μl 5 mM nucleotide mixture

[0120] 5 μl 10X buffer, Promega

[0121] 5 μl MgCl₂, 25 mM

[0122] 0.2 μl (1U) Taq polymerase (5U/μl)

[0123] 31.8 μl H₂ O

[0124] The characteristics of PCR pre-amplification are the following:

[0125] 20 cycles with

[0126] denaturing: 30 sec at 940° C.

[0127] hybridization: 30 sec at 56° C.

[0128] elongation: 1 min at 72° C.

[0129] Selective amplification is made using an aliquot of the firstamplification diluted to {fraction (1/30)} using primers having 3selective nucleotides at the 3′ end, and by labelling one of the primersto develop bands on autoradiography film.

[0130] The following primer pairs are used:

[0131] E-AAC/M-CAG

[0132] E-ACC/M-CAG

[0133] E-AGC/M-CAG

[0134] in which

[0135] E meets the sequence:

[0136] GAC TGC GTA CCA ATT C (SEQ ID N^(o) 1), and

[0137] M meets the sequence:

[0138] GAT GAG TCC TGA GTA A (SEQ ID NO²)

[0139] The hybridization temperature is reduced by 0.7° C. per cycle,throughout the 11 following cycles:

[0140] last 20 cycles

[0141] denaturing: 30 sec at 90° C.

[0142] hybridization: 30 sec at 56° C.

[0143] elongation: 1 min at 72° C.

[0144] The EcoRI primer is labelled (for 0.5 μl tube):

[0145] 0.18 μl EcoRI primer (5 ng)

[0146] 0.1 μl γ³³ P ATP (10 mCu/μl)

[0147] 0.05 μl 10X kinase buffer

[0148] 0.02 μl (0.2u) T4 polymerase kinase (10U/μl)

[0149] 0.15 μl H₂ O

[0150] The labelling reaction is conducted at 37° C. for 1 hour and ishalted by 10 minutes at 70° C.

[0151] c2- Specific amplification reaction

[0152] (20 μl)

[0153] 0.5 μl labelled EcoRI primer

[0154] 5 μl preamplification reaction medium, diluted to

[0155] {fraction (1/30)}

[0156] 0.3 μl MseI primer (100 ng/μl)

[0157] 0.8 μl 5 mM nucleotide mixture

[0158] 2 μl 10X buffer, Promega

[0159] 2 μl MgCl₂, 25 mM

[0160] 0.1 μl (0.5U) Taq polymerase (5U/μl)

[0161] 9.3 μl H₂ O a

[0162] Amplification characteristics are as follows:

[0163] 32 cycles with

[0164] for the first cycle:

[0165] denaturing: 30 sec at 94° C.

[0166] hybridization: 30 sec at 65° C.

[0167] elongation: 1 min at 72° C.

[0168] for the 11 following cycles: the same conditions as previously,reducing the hybridization temperature by 0.7° C. for each cycle; and

[0169] for the 20 last cycles:

[0170] denaturing: 30 sec at 90° C.

[0171] hybridization: 30 sec at 56° C.

[0172] elongation: 1 min at 72° C.

[0173] d) Electrophoresis and Autoradiography

[0174] At the end of the amplification reaction, 20μl of charge bufferare added (98% formamide, 0.005% xylene cyanol and 0.005% bromophenolblue). The amplification products are separated by electrophoresis ondenaturing polyacrylamide gel (6% acrylamide, 8 M urea) with a TBEmigration buffer (18 mM Tris, 0.4 mM EDTA, 18 mM boric acid, pH 8.0) for3 hours' migration at a power of 50 watts. After migration, the gel isfixed in a solution of 1 part acetic acid/ 2 parts absolute ethanol for20 minutes. The gel is transferred to 3 M Wattman paper and dried for 45minutes at 80° C. with a gel drier. The gel is placed in a cassette withultrasensitive film. The autoradiograph is developed after two days'exposure. Comparison of the profiles obtained with the parents and thepools of sensitive of resistant plants led to identifying bands presentin one of the pools but absent in the other. These bands, candidates forresistance marking, were then verified individually on each of theplants forming the DNA pools.

[0175] e) Results

[0176] Study of the results obtained shows that the two markers calledM1 and M2 are present in the sensitive parent (IR64) and in all F2plants (IR64×Gigante) forming the pool of sensitive plants, whereas thisband is absent in the resistant parent (Gigante) and that only oneindividual in the resistant pool shows this band. The same type ofvariation is observed in backcross (IR64×Tog55681)×Tog 5681. The othermarkers identified by this analysis (M3 to M6) also show the samevariation:

[0177] presence of bands in the sensitive parent and the pool of F2sensitive plants (IR64×Gigante) and in the sensitive plants of thebackcross (IR64×Tog5681) ×Tog5681).

[0178] absence of bands in the resistant parents Gigante and Tog5681, inthe pool of F2 resistant plants (IR64×Gigante) and in the resistantplants of the backcross (IR64×Tog5681) ×Tog5681.

[0179] The segregation data between the AFLP markers M1 to M6, theresistance locus for the F2 pools (IR64×Gigante) and the interspecificbackcross (IR64×Tog5681) ×Tog5681 are summarized in tables 4 and 5.Analysis of the segregation data and of the rare recombinants observedin both crosses can be used to assess the recombination rates betweenthese different markers and the resistance locus. In particular, markersMl firstly and markers M2 to M6 secondly determine a segment of lessthan 10-15 cM carrying the resistance locus. M1 and M2 are thereforeless than 5-10 cM apart and are positioned either side of this locus.TABLE 4 Resistance/Marker M1 N° of individuals observed PhenotypeResistant Sensitive RYMV resistance genotype tt/gg tt gg It It It ItAFLP marker −/− +/− +/ −/− +/− −/− +/ Resistant F2 pool 10 — 1 — — — —(IR64 × Gigante) Sensitive F2 pool — — — — — 0 10 (IR64 × Gigante)Interspecific backcross 11 1 — 0 8 — — Tog5681 Resistance/Marker M2, M3,M4, M6 N° of individuals observed Phenotype Resistant Sensitive RYMVresistance genotype tt/gg tt gg It It II II AFLP marker −/− +/− +/ −/−+/− −/− +/ Resistant F2 pool 11 — 0 — — — — (IR64 × Gigante) SensitiveF2 pool — — — — — 0 10 (IR64 × Gigante) Interspecific backcross 10 2 — 08 — — Tog5681 Resistance/Marker M5 N° of individuals observed PhenotypeResistant Sensitive RYMV resistance genotype tt/gg tt gg It It II IIAFLP marker −/ +/− +/ −/− +/− −/− +/ Resistant F2 pool 11 — — — — — 0(IR64 × Gigante) Sensitive F2 pool — — — — — 0 10 (IR64 × Gigante)Interspecific backcross 9 3 0 8 — — — Tog5681

[0180] TABLE 5 Marker M1/Markers M2, M3, M4, M6 N° individuals observedGenotype M1 −/* +/* −/− −/− Genotype M2, M3, M4, M6 +/* −/− +/* −/−Resistant F2 pool 0 1 0 10 (IR64 × Gigante) Sensitive F2 pool 10 0 0 0(IR64 × Gigante) Interspecific backcross 11 2 2 11 Tog5681 MarkerM1/Marker M5 N° individuals observed Genotype M1 −/* +/* −/− −/−Genotype M5 +/* −/− +/* −/− Resistant F2 pool 0 1 0 10 (IR64 × Gigante)Sensitive F2 pool 10 0 0 0 (IR64 × Gigante) Interspecific backcrossTog5681 11 2 3 10 Marker M5/Markers M2, M3, M4, M6 N° individualsobserved Genotyne M5 +/* +/* −/− −/− Genotype M2, M3, M4, M6 +/* −/− +/*−/− Resistant F2 pool 0 0 0 11 (IR64 × Gigante) Sensitive F2 pool 10 0 00 (IR64 × Gigante) Interspecific backcross Tog5681 13 1 0 12

EXAMPLE 5 ISOLATION OF MARKER M1

[0181] A further amplification with the same pair of primers wasconducted, followed by migration on polyacrylamide gel under the sameconditions as above. Development was carried out by staining with silvernitrate using the silver staining kit (Promega) for direct viewing ofthe bands on the gel. After development, the Ml band was excised fromthe gel, then the DNA was eluted in 50 μl water at 4° C. overnight.

[0182] An aliquot of 5 μl was taken and re-amplified using the sameprimer pairs with P³³ labelling. The amplification product was againseparated on 6% denaturing acrylamide gel and compared with the parentsand the sensitive and resistant pools. The lane corresponding to thisamplification product shows a single band of 510 bp migrating at exactlythe same level as the original band which had been excised. Another 5 μlaliquot was also amplified with the same primers and separated on 1.8%agarose gel. The band corresponding to the expected size (510 bp) wasagain excised and purified with a gene clean kit (Promega)

EXAMPLE 6 CLONING AND SEQUENCING OF THE M1 MARKER

[0183] cloning

[0184] 3 μl of purification product was used for a cloning reactionovernight at 37° C.

[0185] 3 μl purification product

[0186] 1 μl PGEMTeasy vector

[0187] 1 μl 10X T4 ligase buffer

[0188] 1 μl T4 DNA Ligase

[0189] 4 μl H₂ O

[0190] Transformation was conducted with the E.Coli strain JM109, adding5 μl of cloning product to 100 μl competent E. coli JM109 cells. Apre-culture was made on LB culture medium for 1 hour at 37° C. Thebacteria were subsequently spread over a Petri dish containing agar with1/1000 ampicilline. 50 μl IPTG-XGal were added just before spreading thebacteria to select the transformed bacteria. A white colony (transformed) was selected and replaced in culture under the sameconditions (Agar plus ampicilline).

[0191] From this culture a miniprep of plasmid DNA was MADE using theWizard Plus kit (Promega) . The plasmid DNA containing the insert wasdigested with the EcoRI enzyme to verify the presence of the Ml marker.1.8% agarose gel was used to verify the presence of the 3 kb bandcorresponding to the plasmid and the 510 bp band corresponding to the M1marker (photo 1).

[0192] Sequencing

[0193] The sequence of the insert (SEQ ID N^(o) 3) is the following(5′,3′): SEQ ID N^(O)3 20      30      40      50      60      70GTGCTTGCTTATAGCACTACAGGAGAAGGAAGGGGAACACAACAGCCATGGCGAGCGAAGGTTCAACGTCGGAGAAACAGGCTGCGACGGGCAGCAAGGTGCCGGCGGCGGATCGGAGGAAGGAAAAGGAGGAAATCGAAGTTATGCTGGAGCGGCTTGACCTAAGGGCAGATGAGGAGGAGGATGTGGAATTGGAGGAAGATCTAGAGGAGCTTGAGGCAGATGCAAGATGGCTAGCCCTAGCCACAGTTCATACGAAGCGATCGTTTAGTCAAGGGGCTTTCTTTGGGAGTATGCGCTCAGCATGGAACTGCGCGAAAGAAGTAGATTTCAGAGCAATGAAGACAATCTGTTCTCGATCCAATTCAATTGTTTGGGGGATTGGAACGAGTTATGAATGAAGGTCCATGGACCTTTCGAGGATGTTCGGTGCTCCTCGCAGAATATGATGGCTGGTCCAAGATTGAAT

[0194] The sequences corresponding to the primers used for AFLPamplifications were found and show that the band corresponds to arestriction fragment (EcoRI-MseI).

[0195] By deducing the sequences corresponding to the primers, theactual size of the DNA fragment of the cloned rice is 471 bp.

[0196] The use of different pairs of primers (1-3), (1-4), (1-5) firstlyand (2-3), (2-4), (2-5) secondly, makes it possible to validate thecloning of the AFLP M1 band. Amplification of the DNA of the varietiesused in the crosses with these primers only shows one single band. Thefragment corresponding to the Tog5681 variety is slightly larger thanfor the other varieties (FIG. 2).

EXAMPLE 7 TRANSFORMATION OF THE M1 SEQUENCE INTO A POLYMORPHOUS MARKER

[0197] A polymorphism for the Ml marker was determined between theparents of the doubled haploid population (IR64×Azucena) . Thispopulation totals over 300 markers distributed over the 12 ricechromosomes. On this account, we relied on the restriction sites of theM1 marker sequence determined on the IR64 parent (FIG. 3). The primers(1-3), (1-4) and (1-5) were used to amplify the DNA of the parents ofcrossed plants which was then digested by restriction enzymes. Therestriction site HpaII/MspI releases a fragment of 86 bp when primer 1is used. This site is absent in the Gigante and Azucena varieties (FIG.4).

[0198] The marker was tested on the F2 individuals of the sensitive pooland resistant crossed pool (IR64×Gigante). All the resistant individualshave the profile of the Gigante variety (absence of the M1 AFLP markerassociated with absence of the restriction site HpaII/MspI) with theexception of individual (5.11). The sensitive individuals show theHpaII/MspI restriction site in the homozygote state like the IR64variety with the exception of two heterozygote individuals which arerecombinant (FIG. 5).

[0199] The sequence of the M1 marker which can be amplified withspecific primers indeed corresponds to the M1 AFLP marker. Digestion bythe HpaII/MspI enzyme leads to distinguishing between the allele derivedfrom the sensitive parent (IR64) and from the resistant parent(Gigante).

[0200] With these new data, it is possible to give back-up to thepositioning of the resistance locus between markers M1 and M2 and toestimate the recombination rate at 0.065 ±0.045 for the distance betweenM1 and the resistance locus, and 0.11 ±0.047 for the distance betweenmarkers M1 and M2.

EXAMPLE 8 MAPPING OF THE M1 MARKER

[0201] Sixty individuals from the (IR64×Azucena) population were passedas marker M1: amplification with primers (1-3) and digestion with theHpaII/MspI enzyme, followed by separation of the fragments on 2.5 %agarose gel. Segregation of marker M1 shows no distortion (FIG. 6) . Theresults are used to map the Ml marker using mapping software (MapmakerV3) which leads to positioning the M1 marker on chromosome 4 between themarkers RG163 and RG 214(FIG. 7) . This space represents the zone inwhich the RYMV resistance locus is located.

EXAMPLE 9 MARKING THE RESISTANCE LOCUS OF THE TOG5681 VARIETY

[0202] The presence of the restriction site HpaII/MspI in the Tog5681variety means that it is not possible to use the strategy in example 8to verify that the Ml marker is also a marker of Tog5681 resistancederived from Tog5681. Therefore, the 4 varieties Azucena, Gigante, IR64and Tog5681 were digested with 12 restriction enzymes (BamHI, Bg/II,DraI, EcoRI, EcoRV, HindlIl, Apal, KpnI, PstI, Scal, XbaI, HaeIII) toidentify a restriction polymorphism using the DNA sequence of the Mlmarker as probe. The Scal enzyme leads to identifying a polymorphismbetween IR64 and Tog5681(FIG. 8). This polymorphism was used to validatethe M1 marker on a backcross (IR64×Tog5681) ×IR64 in segregation forresistance. 5 sensitive individuals of this backcross were tested andall showed the characteristic band of IR64. The 9 resistant individualsonly show the Tog5681 band with the exception of only one which isrecombinant (FIG. 9). The restriction polymorphism revealed by the Scalenzyme using the M1 marker as probe is therefore related to theresistance locus of Tog5681. There is coherence between genetic analysisand the identification of resistance markers for considering that the M1marker indeed maps the same resistance locus in the two varietiesGigante and Tog5681.

EXAMPLE 10 CLONING AND SEQUENCING OF THE M2 MARKER INTO A SPECIFICPCR-MARKER

[0203] The AFLP band obtained with the pair of primers E-ACC/M-CAGcorresponding to the M2 band visible in the sensitive parent (IR64) andpresent in all the individuals forming the sensitive pool, was clonedusing the same protocol as for marker M1. The sequence corresponding tothis band was determined and 3 primers were defined (1 forward - 2reverse) to allow conversion of this marker into a specific PCR marker.Sequence of the M2 marker (120 bp) (SEQ ID NO.⁹): AATTCACCCC ATGCCCTAAG TTAGGACGTT CTCAGCTTAG TGGTGTGGTA GCTTTTTCTA TTTTCCTAAG CACCCATTGAAGTATTTTGC ATTGGAGGTG GCCTTAGGTT TGCCTCTGTTA Primers:AACCTAAGGCCACCTCCAAT: (right) (SEQ ID NO 10) GCAAACCTAAGGCCACCTC:(right) (SEQ ID NO 11) ATTCACCCCATGCCCTAAG: (left) (SEQ ID NO 12)

[0204] The following conditions were used to amplify markers M1 and M2simultaneously: 10 X buffer, Promega 1.5 μl MgCl₂ Promega 1.5 μl dNTP (5mM) 0.6 μl M1-1 primer (10 mM) 0.15 μl M1-4 primer (10 mM) 0.15 μl M2-1primer (10 mM) 0.15 μl M2-2 primer (10 mM) 0.15 μl H₂O 7.74 μl TaqPolymerase 0.06 μl DNA (5 ng/μl) 3.00 μl

[0205] PCR programme:

[0206] 5 min at 94° C.

[0207] 1 mn at 94° C.

[0208] 30 s at 59° C.

[0209] 1 mn at 72° C.

[0210] 35 cycles

[0211] 5 mn at 72° C.

[0212] 10 mn at 4° C.

[0213] The M2 marker may be amplified alone at a hybridizationtemperature of 60.5° C., the other parameters remaining unchanged. Underthese amplification conditions, the M2 marker appears to be a dominantmarker characterized by band presence in the sensitive parent (IR64) andband absence in the Gigante parent.

EXAMPLE 11 Creation of a population of recombinant resistant plantsbetween markers M1 and M2 to arrange within this space the candidateAFLP markers for resistance marking.

[0214] 750 F2 individuals (IR64×Gigante) were artificially inoculatedwith the RYMV virus (BFl strain). The symptom-free plants weretransplanted to a greenhouse, i.e. 188 individuals. Subsequently,additional analysis based on ELISA and descendant tests made it possibleto eliminate a last fraction of 50 sensitive plants. The remaining 138plants, homozygote for resistance, were systematically genotyped forboth markers M1 and M2 as previously described. In this manner, 45individuals were selected (38 recombinant relative to M1. 7 recombinantrelative to M2) and 2 double recombinants. These recombinant individualswere used for arranging the AFLP markers in the space between M1 and M2.These results are summarized in Table 6 below: TABLE 6 Selection of arecombinant F2 sub-population (IR64 x Gigante) in the M1-M2 marker spaceN° of Steps conducted: F2 (IR64 × Gigante) plants % Inoculation of F2plants (10 days after sowing) 768 Greenhouse transplantation (5 weeksafter 188 inoculation) Elimination of sensitive plants 50 (symptomfollow-up - Elisa test, descendant test) Selection of homozygoteresistant plants for the 138 17.9 bred resistance gene Genotyping ofselected individuals for markers M1 and M2 Recombinant plants relativeto M1 36 18.8 Recombinant plants relative to M1 and M2 2 1.4 Recombinantplants relative to M2 7 5.1

EXAMPLE 12 Screening of AFLP markers to select new candidate markers forresistance

[0215] A total of 328 primer pairs EcoRI/MseI, each one defined by 3nucleotides, was used following the protocol previously described. Theseprimers are given in Table 7 below. TABLE 7 Combination EcoRI MesICombination EcoRI MseI Combination EcoRI MseI NO primer primer NO primerprimer NO primer   1 AAC CAA  55 ACA CTG 109 ACG AGG   2 AAC CAC  56 ACACTT 110 ACG ACT   3* AAC CAG  57 ACA AAC 111 ACT CAA   4 AAC CAT  58 ACAAAG 112 ACT CAC   5 AAC CCA  59 ACA AAT 113 ACT CAC   6 AAC CCT  60 ACAACA 114 ACT CAT   7 AAC CGA  61 ACA ACG 115 ACT CCA   8 AAC CGT  62 ACAACG 116 ACT CGT   9 AAC CTA  63 ACA ACT 117 ACT CGA  10 AAC CTC  64 ACAAGC 118 ACT CGT  11 AAC CTG  65 ACA AGG 119 ACT CTA  12 AAC CTT  66 ACAAGT 120 ACT CTC  13 AAC AAC  67 ACC CAA 121 ACT CTG  14 AAC AAG  68 ACCCAC 122 ACT CTT  15 AAC AAT  69* ACC CAG 123 ACT AAC  16 AAC ACA  70 ACCCAT 124 ACT AAG  17 AAC ACC  71 ACC CCA 125 ACT AAT  18 AAC ACG  72 ACCCCT 126 ACT ACA  19 AAC ACT  73 ACC CCA 127 ACT ACC  20 AAC AGC  74 ACCCGT 128 ACT ACG  21 AAC ACG  75 ACC CTA 129 ACT ACT  22 AAC ACT  76 ACCCTC 130 ACT AGC  23 AAC CAA  77** ACC CTG 131 ACT AGG  24 AAG CAC  78ACC CTT 132 ACT AGT  25 AAG CAG  79 ACC AAC 133 AGA CAA  26 AAG CAT  80ACC AAG 134 AGA CAC  27 AAG CCA  81** ACC AAT 135 AGA CAG  28 AAG CCT 82 ACC ACA 136 ACA CAT  29 AAG CGA  83 ACC ACC 137 AGA CCA  30 AAG CGT 84 ACC AGG 138 AGA CCT  31 AAG CTA  85 ACC ACT 139 AGA CGA  32 AAC CTC 86** ACC AGC 140 AGA CGT  33 AAG CTG  87 ACC AGG 141 AGA CTA  34 AACCTT  88 ACC AGT 142 AGA CTC  35 AAG AAC  89 ACG CAA 143 ACA CTG  36 AAGAAG  90 ACG CAC 144 ACA CTT  37 AAG AAT  91** ACG GAG 145 AGA AAC  38AAG ACA  92 ACG CAT 146 AGA AAG  39 AAG ACC  93 ACG CCA 147 AGA AAT  40AAG ACG  94 ACG CCT 148 AGA ACA  41 AAC ACT  95 ACC CGA 149 AGA ACC  42AAG AGC  96 ACG CCT 150 ACA ACG  43 AAG ACG  97 ACG CTA 151 AGA ACT  44AAG AGT  98 ACG CTC 152 ACA ACC  45 ACA CAA  99 ACG CTC 153 AGA AGG  46ACA CAC 100 ACG CTT 154*** ACA ACT  47 ACA CAC 101 ACC AAC 155 AGC CAA 48 ACA CAT 102 ACG AAC 156 ACC CAC  49 ACA CCA 103 ACG AAT 157*** ACCCAG  50 ACA CCT 104* ACG ACA 158 AGC CAT  51 ACA CCA 105 ACG ACC 159 AGCCCA  52 ACA CGT 106 ACG ACG 160 AGC CCT  53 ACA CTA 107 ACG ACT 161 ACCCGA  54 ACA CTC 108 ACG ACC 162 ACC CGT Shaded: polymorphism for one ormore bands between the sensitive and re- sistant pools * presence of oneor more polymorphous bands in sensitive pool ** presence of one or morepolymorphous bands in resistant pool *** presence of one or morepolymorphous bands in sensitive pool and re- sistant pool 163 AGC CTA218 AGT AGC 273 CAT CTA 164 AGC CTC 219 ACT ACG 274 CAT CTC 165 AGC CTC220* AGT ACT 275 CAT CTG 166 ACC CTT 221 ATC CAA 276 CAT CTT 167 ACC AAC222 ATC CAC 277 CAT AAC 168 AGC AAC 223 ATC CAG 278 CAT AAG 169 AGC AAT224 ATC CAT 279 CAT AAT 170 ACC ACA 225 ATC CCA 280* CAT ACA 171 AGC ACC226 ATC CCT 281 CAT ACC 172 AGC ACG 227 ATC CGA 282 CAT ACG 173 AGC ACT228 ATC CGT 283 CAT ACT 174** AGC AGC 229 ATC CTA 284 CAT ACC 175*** AGCAGG 230 ATC CTC 285 CAT AGG 176 AGC ACT 231 ATC CTG 286 CAT AGT 177 AGCCAA 232 ATC CTT 287* ACT CAA 178 AAC CAC 233*** ATC AAC 288 CTA CAC 179AGG CAG 234*** ATC AAG 289 CTA CAG 180 AGG CAT 235* ATC AAT 290 CTA CAT181 AGG CCA 236 ATC ACA 291* CTA CCA 182 AGG CCT 237 ATC ACC 292 CTA CCT183 AGG CGA 238 ATC ACG 293 CTA CCA 184 AGG CGT 239 ATC ACT 294 CTA CGT185 AGG CTA 240 ATC AGC 295 CTA CTA 186 AGG CTC 241 ATC AGG 296 CTA CTC187 AGG CTG 242 ATC ACT 297* CTA CTG 188 AGG CTT 243 CAA CAA 298 CTA CTT189 AGG AAC 244 CAA CAC 299 CTA AAC 190 AGG AAG 245 CAA CAG 300 CTA AAG191 AGG AAT 246 CAA CAT 301 CTA AAT 192 AGG ACA 24i CAA CCA 302 CTA ACA193 AGG ACC 248 CAA CCT 303 CTA ACC 194 AGG ACG 249 CAA CGA 304 CTA ACG195** AGG ACT 250** CAA CGT 305 CTA ACT 196 AGG AGC 251 CAA CTA 306 CTAAGC 197*** AGG AGG 252 CAA CTC 307 CTA AGG 198 AGG ACT 253 CAA CTG 308CTA ACT 199 ACT CAA 254* CAA CTT 309 CTT CAA 200 ACT CAC 255 CAA AAC 310CTT CAC 201 ACT CAG 256 CAA AAG 311 CTT CAG 202 ACT CAT 257* CAA AAT312** CTT CAT 203 ACT CCA 258** CAA ACA 313 CTT CCA 204 AGT CCT 259 CAAACC 314 CTT CCT 205 AGT CGA 260 CAA ACG 315 CTT CGA 206 ACT CGT 261 CAAACT 316 CTT CGT 207 ACT CTA 262 CAA AGC 32.7 CTT CTA 208 ACT CTC 263 CAAAGG 318* CTT CTC 209 AGT CTG 264 CAA AGT 319** CTT CTG 210 AGT CTT 265CAT CAA 320 CTT CTT 211 AGT AAC 266 CAT CAC 321 CTT AAC 212 AGT AAG 267CAT CAG 322 CTT AAG 213* AGT PAT 268 CAT CAT 323 CTT AAT 214 AGT ACA 269CAT CCA 324 CTT ACA 215** AGT ACC 270 CAT CCT 325 CTT ACC 216 AGT ACG271 CAT CGA 326 CTT ACG 217 AGT ACT 272* CAT CGT 327 CTT ACT 328 CTT AGT** presence of one or more polymorphous bands in resistant pool ***presence of one or more polymorphous bands in sensitive pool andresistant pool

[0216] With this screening, it was possible to identify one or morepolymorphous bands according to their occurrence in the sensitive parentand/or resistant parent. 23 primer pairs were able to identifypolymorphism between the parents confirmed by the F2 DNA pools,sensitive or resistant. The table below summarizes and gives theposition in the M1-M2 space of AFLP markers bound to the locus of bredresistance the rice yellow mottle virus. TABLE 8 Presence of Variableband(s) Combi- nucleotides Sensi- Resis- nation EcoRI MseI tive tantMarker position Number primer primer pool pool in M1-M2 space 3 AAC CAG− − =cloned M1 marker 69 ACC CAG + − =cloned M2 marker 77 ACC CTG − +non-determined 81 ACC AAT − + non-determined 86 ACC AGC − +non-determined 91 ACG − + non-determined 104 ACG ACA + − betw R andRm273 154 ACA ACT + + beyond M2 157 AGC CAG − + in cosegr with M2 174AGC AGC − + non-determined 175 AGC ACG + + betw M1 and Rm241 197 AGCAGG + − betw M1 and Rm241 215 AGT ACC − + non-determined 220 ACT AGT + −betw Rm273 and M2 233 ATC AAG + + betw M1 and Rm241 250 CAA CGT − +non-determined 254 CAA CTT + − beyond M2 258 CAA ACA + − betw M1 andRm241 280 CAT ACA + − beyond M2 287 CTA CAA + − betw Rm273 and M2 291GTA CCA + − betw M1 and Rm241 318 CTT CTC + + bewt Rm273 and M2 319 CTTCTG − + non-determined

[0217] After separate verification on each of the individuals formingthe pools, the candidate markers corresponding to bands present in theIR64 parent may be tested on the recombinants identified in example 11.In this manner, 9 markers were confirmed as belonging to the M1-M2space. Table 9 gives the order in the M1-M2 space of the AFLP markersidentified by comparing sensitive and resistant DNA pools from aresistant F2 sub-ion (IR64×Gigante). TABLE 9 F2 Resistant E− E− E− E− E−E− E− C− E− individuals AGG ATC CAA AGC CTA ACG AGT CTT CTA (IR64× M− M−M− M− M− RYMV M− M− M− M− Gigante) M1 AGG AGG ACA AGG CCA RM241 RM252resist ACA RM273 AGT CTC CAA M2 2 H D D D D D B B B B B B B B 7 H D D DD D B B B B B B B B 8 H D D D D D B B B B B B B B 10 H D D D E D B B B BB B B B 21 H D D D D B C B B B B B B B B 23 H D D D E D B B B B B B B B25 H D D D D D E H B B B B B B 28 H D D D B B C B B B B B B B B 37 H D DD E D E H B B B B B B B 48 H D D D D D B B B B B B B B 55 H D D D D D EH B B B B B B B 61 H D D D D D E H B B B B B B B 65 H D D D B C B B B BB B B B 95 H E D D D B C B B B B B B B B 103 H E D D B C B B B B B B B B104 H D D D B B C B B B B B B B B 109 H D D D D D C B B B B B B B B 111H E D D D D B B B B B B B B 119 H D D D D D B B B B B B B B 120 A D D DD B C B B B B B B B B 125 H E E E E D B B B B B B B B 127 H B C B B B BB B B B 131 H E E E E D B B B B B B B B 133 H B C B B B B B B B B 141 HE E E E D E H B B B B B B B 154 H E E E E D B B B B B B B B 158 H E E EE D B B B B B B B B 159 H B C B B B B B B B B 160 H E E E E D B B B B BB B B 151 H E E E E D B B B B B B B B 153 H B C B B B B B B B B 157 H BB B B B B B B B B 171 H B B B B B B B B B B 175 H E E E D D B B B B B BB B B 179 H B B B B B B B B B B B 183 H E E E E D B B B B B B B B B 35 HD D D D D H H B D H D D D D 135 H E E E E D H H B B H D D D D 17 H B B BB B B B D H D D D D 20 B B B B B B B B B D H D D D D 38 B B B B B B B BD H D D D D 93 B B B B B B B B B D H D D D D 105 B B B B B B B B B D H DD D D 145 B B B B B B B B B B B D 180 B B B B B B B B B D D D D

[0218] M1-R space 0.97 0.97 0.97 087 0.61 0.29 0.13

[0219] R-M2 space 0.67 0.78 0.89 0.89 0.89

[0220] Distance/resistance (cM) 11.4** 11.03 11.03 11.03 9.88 6.90 3.332.10 0.00 3.33 3.89 4.44 4.44 4.44 5.0**

[0221] A: genotype homozygote for the allele of the sensitive parent(IR64)

[0222] H: heterozygote genotype

[0223] B: homozygote genotype for the allele of the resistant parent(Gigante)

[0224] D: genotype non homozygote for the allele of the resistant parent(Gigante)

[0225] under the assumption of absence of double combination in spaceM1-R and M2-R

[0226] estimated distance using resistance map on 183 F2 (IR64×Gigante)cf (figure X)

[0227] 14 bands from the resistant parent were also identified and willor will not be confirmed on recombinants generated in the F2 population(IR64×Gigante).

EXAMPLE 13 Anchoring of the RYMV resistance locus using microsatellitemarkers

[0228] The M1 marker being positioned on chromosome 4 of the genetic map(IR64×Azucena; example 9) microsatellite markers such as defined in (6)and belonging to this chromosome were used to fine-tune the map of theRYMV resistance locus. The following microsatellite markers were tested:RM241, RM252 (1), RM273 and RM177(6), under the experimental conditionsdescribed in (1) and (6). With the exception of the RM177 marker,non-polymorphous between the IR64 and Gigante parents, the markersRM241, RM252, RM273 were mapped on a F2 population (IR64×Gigante)assessed in parallel for RYMV resistance. The results on 183 F2individuals make it possible to characterized a stretch of approximately3.6 cM bordered by the two microsatellite loci RM252 and RM272surrounding the RYMV resistance gene (see FIG. 10(a)).

EXAMPLE 14 Fine mapping of the space carrying the resistance locus andorder of the resistance markers in the M1-M2 space.

[0229] The 45 F2 individuals (IR64×Gigante) resistant and recombinantfor the M1 and m2 markers were characterized for the microsatellitemarkers identified in example 13. The mapping of the markers insegregation on all the F2 individuals (IR64×Gigante) available (321)confirms the order and the distance between the markers of the M1-M2space, in particular the RM252-RM273 space which is estimated at 3.6 cM(FIG. 10(b)). With the 45 F2 individuals (IR64×Gigante) that areresistant and recombinant for the M1 and M2 markers, it is possible toconfirm the order of the AFLP markers identified in example 12. One AFLPmarker, EACG/MACA, remains within the RM252-RM273 space and representsthe nearest marker to the RYMV resistance locus (Table 9). Overall, outof the 321 F2 individuals tested, there are 20 individuals recombined onone side or other of the RYMV resistance locus and may advantageously beused to identify closer markers and/or for cloning the resistance gene.

EXAMPLE 15 Marker-assisted resistance transfer

[0230] The markers close to the resistance locus were tested onirrigated varieties highly sensitive to the RYMV virus (var BG90-2,Bouake189, Jaya). 3 markers (M1, RM241, RM252) show polymorphism betweenthese 3 varieties and the Gigante variety, enabling the use of thesemarkers to be considered for resistance transfer to sensitive genotypes.Experimental transfer of resistance to these varieties was made as faras the 2 ^(nd) backcross. At each cross, the plants were verified forthe presence of markers derived from Gigante, and resistance segregationwas controlled by descendant tests on F2. Table 10 below summarizesresults. TABLE 10 theoretical Polymorphism/donor parent % N° ofRecurrent (Gigante) Generation recurrent lines parent M1 RM241 RM252RM273 RM177 obtained parent obtained BG90-2 poly poly poly — — BC2F287.5 4 Bouaké poly poly poly — — BC2F2 87.5 1 189 poly poly poly — —BC2F2 87.5 2 Jaya poly poly poly poly mono BC3 93.7 5 IR64

EXAMPLE 16 Anchoring of the resistance gene on the physical map

[0231] The AFLP band corresponding to the M3 marker and amplified in thesusceptible parent IR64 with the primers E-ACG/M-ACA has been clonedusing the same conditions than for Ml marker. This band was sequenced:

[0232] Sequence of M3 marker (excluded adaptators):Acggacctatccacttttatgccagcaagaaaatttagatgatggcaactgtatg t (seq. N^(o)13)

[0233] DNA from varieties Gigante, IR64, Azucena and Tog5681 wasdigested using restriction enzymes Hind III, Eco RV, Dra I, Xba I, BglII, Bam HI, Sca I et Eco RI and membranes has been realized.Hybridization of the M3 sequence on these membranes did not revealpolymorphism between tested varieties. However, hybridization profilerevealed that M3 is a single copy sequence in rice genome. This probehas been used to screen a BAC library including 36000 clones, realizedin Clemson University using DNA of Nipponbare variety, digested withHind III enzyme.

[0234] Membrane prehybridization was performed one night at 65° C. inhybridization tubes, in a buffer made of SDS 7%, sodium phosphate 0.5MpH7.2, EDTA 1 mM, salmon sperm DNA (0.1 mg/ml). Hybridization wasperformed in the same buffer in which labeled probe was added. Probe wasradioactively labeled using the “5′-end-labelling” kit fromAmersham-Pharmacia, as recommended by furnisher. After one night at 65°C., membranes were washed twice 20 minutes in SSC 1 X, SDS 0.1% andtwice 20 minutes in SSC 0.5X, SDS 0.1%. The, membranes were wrapped inSaran-wrap and kept at −80° C. in contact with film.

[0235] Probe corresponding to M3 marker hybridized on 17 clones, 13 ofwhich belong to contig 89, as described on Clemson University web site () on JUN. 12, 2001. These clones were : OSJNBa0006L19, OSJNBa0015F04,OSJNBa0022014, OSJNBa0032M10, OSJNB a0048E10, OSJNBa0043I12,OSJNBa0051M11, OSJNBa0052K13, OSJNBa0059I01, OSJNBa0058F05,OSJNBa0070I17, OSJNBa0083D09. OSJNBa0087J22. These results are coherentenough to consider that M3 is on contig 89. A figure of the contig 89 isgiven (FIG. 11), clones hybridizing with M3 are indicated using a thicktrait.

EXAMPLE 17 Position of the gene on contig 89

[0236] Several BAC of contig 89 have been sequenced in the internationalrice genome sequencing project and sequenced were released on databanks. Thus, the BAC clone OSJNBa0014 K14, localized, at one extremityof contig 89, has been sequenced and its sequenced has been recordedunder accession number AL606604 on GenBank. A microsatellite sequencewas identified on this clone and primers have been designed on bothsides of this sequence in order to develop a microsatellite marker(referred later as MS606604-2).

[0237] Microsatellite sequence (upper case) and primers designed inflanking sequences (underlined):GcaaagtgtttcaccttggacccatgcattCCTCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCgagcgctcaactctccattgagcactgagcaggcccttacctttgcct Gcaaagtgtttcaccttggacc (sequence NB 14)Agcaggcccttacctttgcct (sequence NB 15)

[0238] This marker was amplified on varieties IR64, Gigante, Nipponbareand Tog5681 in the following conditions: dNTP 200 μM Taq 0.02 U/μlBuffer 1X MgCl2 1.5 mM Forward primer-M13 0.1 μM Reverse primer 0.1 μMPrimer M13-IRD700 0.06 μM (amplification in 15 μl)

[0239] In order to visualize amplification products on a LICORsequencer, amplification is performed using the M13-forward universalprimer labeled with IRD700 and the forward MS606604-2 primer to whichthe sequence 13-forward is added in 5′ position (tailing protocoldescribed by furnisher). Amplification is realized with the program:

[0240] 5 min 94° C.

[0241] 30 s 94° C.

[0242] 30 s 57° C.

[0243] 5 40 s 72° C.

[0244] (34 cycles)

[0245] 5 min 72° C.

[0246] A size-based polymorphism was detected between IR64 and Gigantevarieties. This marker has been tested on 30 individuals recombinedbetween RM252 and RM273 (12 resistant plants already presented in table9 and 18 additional individuals evaluated for resistance level on F3progenies). The marker MS606604-2 showed a perfect co-segregation withRM252 (table 11) TABLE 11 RYMV M1 RM241 MS606604-2 RM252 resistance M7RM273 M2 Resistant F2 plants recombined between RM252 and RM273 F2-R17 B— B B B D H D F2-R20 B B B B B D H D F2-R25 H — H B B B B F2-R36 H H H HB D H D F2-R37 H — H H B B B B F2-R38 B — B B B D H D F2-R55 H — H B B BB F2-R61 H — H H B B B B F2-R93 B B B B B D H D F2- B B B B B D H D R105F2- H H H H B B H D R135 F2- H — H H B B B B R141 F2 plants recombinedbetween RM252 and RM273, and evaluated for resistance on F3 progeniesBR5 (11) H H H B B B B F2-1 H H H H H B B B F2-16 H H A A A D H D F2-19H B B B H — H D F2-95 A A A A H D H D F2-113 H — H H H B B B F2-114 — HH H B — H F2-133 H A A A H D H D F2-142 H — H H B B B B F2-163 B — B B BD H D F2-167 B — H H H D A D F2-176 A A A A A D H D F2-184 — — H H H D AD F2-189 H — H H H D B B F2-206 — H H H H B B F2-223 — A A A H D HF2-278 B — B B B — H D F2-280 H A A A A D H D F2-285 — — A H — H

[0247] Resistance gene is localized between markers M3 and MS606604-2and thus between the position delimited by these markers on contig 89,as mentioned on FIG. 11.

BIBLIOGRAPHIC REFERENCES

[0248] (1) Chen, X et al., (1997), Development of a microsatelliteframework map providing genome-wide coverage in rice (Oryza saliva L)Theor Appl Genet 95: 553-567.

[0249] (2) Panaud, 0. et al., (1996), Development of microsatellitemarkers and characterization of simple sequence length polymorphism(SSLP) in rice (Oryza sativa L) Mol Gen Genet 252: 597-607.

[0250] (3) Wu K.S. et al., (1993), Abundance, polymorphism and geneticmapping of microsatellites in rice. Mol Gen Genet 241: 225-235.

[0251] (4) Zabeau et al., (1993), Selective restriction fragmentamplification: a general method for DNA fingerprinting. EP 92402629.7.

[0252] (5) Vos et al., (1995), AFLP, a new technique for DNAfingerprinting. Nucleic Acids research 23: 4407-4414.

[0253] (6) Temnyck et al., (2000), Theor Appl Genet 100:697-712.

[0254] The entire content of all reference cited or referred to hereinis incorporated herein by reference.

1. Method for identifying markers of the locus of a major resistancegene to RYMV, comprising: selective amplification of rice DNA fragmentsfirstly from resistant individuals, and secondly from sensitiveindividuals, descending from parental varieties, these fragments beingpreviously subjected to a digestion step, then a ligation step to fixcomplementary primer adapters having at their end one or more specificnucleotides, one the primers of the pair being labelled for developmentpurposes, separation of the amplification products, by gelelectrophoresis under denaturing conditions, and comparison of theelectrophoresis profiles obtained with mixtures of fragments derivedfrom resistant descendants and mixtures derived from sensitivedescendants, with fragments derived from parental varieties, for thepurpose of identifying bands whose polymorphism is genetically linked tothe resistance locus, this identification optionally being followed, forvalidation purposes, by verification on each individual and calculationof the genetic recombination rate between the marker and the resistancelocus.
 2. Method according to claim 1, characterized in that the DNAfragments are obtained by digestion of the genomic DNA of resistantplants and of sensitive plants, and their parents, using restrictionenzymes.
 3. Method according to claim 2, characterized in that asrestriction enzymes EcoRI and MseI are used.
 4. Method according toclaim 2 or 3, characterized in that the restriction fragments aresubjected to ligation to fix adapters.
 5. Method according to claim 4,characterized in that the fragments obtained are amplified using primerpairs complementary to the adapters whose sequences are respectively GACTGC GTA CCA ATT C(SEQ ID N^(o) 1) and GAT GAG TCC TGA GTA A(SEQ ID N^(o)2).
 6. Method according to claim 4 or 5, characterized in that thefragments obtained are amplified using primer pairs having at their endthe respective motifs AAC and CAG, ACC and CAG or further AGC and CAG.7. Method according to any of claims 1 to 6, characterized by theidentification of resistance marker bands, M1 and M2, whose size isrespectively 510 bp and 140 bp, such as determined by gelelectrophoresis under denaturing conditions.
 8. Method according toclaim 7, characterized in that said marker bands determine a segment ofless than 10-15 cM carrying the resistance locus.
 9. Method according toclaim 8, characterized in that said marker bands are located either sideof the locus at less than 5-10 cM.
 10. Method according to any of claims1 to 9, characterized in that it also comprises an isolation step toisolate the identified marker bands.
 11. Method according to claim 10,characterized by purification of the isolated marker bands in order toobtain DNA fragments.
 12. Method according to claim 11, characterized bycloning of the marker bands into a vector and insertion of the vector ina host cell.
 13. Method according to either of claims 11 or 12,characterized by the recovery and sequencing of the purified, cloned DNAfragments.
 14. Method for obtaining markers having high specificity forthe locus of a major RYMV resistance gene, characterized in that PCRprimer pairs are defined complementary to the sequence of the clonedfragment, specific amplification of this fragment is carried out usingthese primer pairs, then the amplification products are subjected tomigration on electrophoresis gel with or without previous digestion by arestriction enzyme to identify a polymorphism.
 15. Polymorphous AFLPbands such as identified by the method according to any of claims 1 to14 using rice plant DNA.
 16. AFLP bands according to claim 15,characterized in that they are specifically evidenced in aRYMV-sensitive variety, and in the fraction of sensitive plants derivedfrom crossing of this variety with a resistant variety.
 17. DNAsequences corresponding to polymorphous bands according to claim 15 or16, which can be used to define a segment of chromosome 4 of 10-15 cMcarrying the RYMV resistance locus.
 18. DNA sequences according to claim17, characterized in that they correspond to EcoRI-MseI fragments. 19.DNA sequences according to claim 18, characterized by a respective sizeof 510 bp and 140 bp determined by gel electrophoresis under denaturingconditions.
 20. DNA sequences according to any of claims 17 to 19,characterized in that they correspond to sequences flanking theresistance locus and located either side of the latter at 5-10 cM oreven at less than 5 cM.
 21. DNA sequence, characterized in that it meetsSEQ ID N^(o)
 3. 22. DNA sequence, characterized in that it meets SEQ IDN^(o)
 9. 23. Cloning vectors, characterized in that they containsequence SEQ ID N^(o) 3 according to claim 21 or sequence SEQ ID N^(o) 9according to claim
 22. 24. Host cells, characterized in that they aretransformed by vectors according to claim
 22. 25. Use of polymorphousbands according to claim 15 or 16 or of DNA sequences according to anyof claims 17 to 22 for the identification of resistant phenotypes andtransfer of the resistance gene.
 26. Fragments of no more than 4-5cM ofchromosome 4 and polymorphous AFLP bands according to claim 15 or 16defining a segment of 4-5cM or less carrying the RYMV resistance locus.27. Use of SEQ ID NO.³ or SEQ ID NO.⁹ or of microsatellite markers suchas RM252 and RM273, or any other marker such as SEQ ID NO.¹³ of contig89 of Nipponbare BAC library, and showing polymorphism between asensitive variety and a resistant variety, to transfer resistance into asensitive variety by marker-assisted selection.
 28. Use of sequences ofcontig 89 of Nipponbare library for identifying the sequences of thegene responsible for resistance to the rice yellow mottle virus.